Strained Ammonium Precursors for Radiofluorinations

Abstract The increasing application of positron emission tomography (PET) in nuclear medicine has stimulated the extensive development of a multitude of novel and versatile techniques to introduce fluorine‐18, especially for the radiolabelling of biologically or pharmacologically active molecules. Taking into consideration that the introduction of fluorine‐18 (t1/2=109.8 min) mostly proceeds under harsh conditions, radiolabelling of such molecules represents a challenge and is of enormous interest. Ideally, it should proceed in a regioselective manner under mild physiological conditions, in an acceptable time span, with high yields and high specific activities. Special attention has been drawn to 2‐fluoroethyl and 3‐fluoropropyl groups, which are often the active sites of radiofluorinated compounds. Precursors containing an ammonium leaving group – such as a strained azetidinium or aziridinium moiety – can help to overcome these obstacles leading to a convenient and mild introduction of [18F]fluoride with high radiochemical yields.


Introduction
The positron emitter fluorine-18 is the most and widely used radionuclide for the production of radiopharmaceuticals for positron emission tomography (PET) apart from further shortlived nonmetallic radionuclides carbon-11, nitrogen-13, oxygen-15, [1][2][3] and iodine-124 [4,5] as well as, for example, the radiometals gallium-68, copper-64, scandium-43/-44 and zirconium-89. [6] Special attention has to be paid to the method of the [ 18 F]fluoride introduction. Commonly known radiolabelling methods like nucleophilic and electrophilic substitutions, metal catalyst mediated mechanisms, ligand exchange or prosthetic groups are widely used to regioselectively achieve high radiochemical yields. [7,8] Normally, the introduction of fluorine-18 proceeds via two main routes: the electrophilic way is based on the use of [ 18 F]F 2 , [9] mostly with low molar activities. In contrast, [ 18 F]fluoride is used [10] for the nucleophilic way, typically with high molar activities of the resulting radiotracer. Usually, nocarrier-added [ 18 F]fluoride is applied for nucleophilic reactions with high molar activity in form of its K[ 18 F]F-K222 (Kryptofix) complex, proceeding as S n 2-type substitutions for aliphatic moieties and as S N Ar-type substitutions for (hetero)aromatic moieties. [11] Appropriate leaving groups such as halogens or sulfonates are required for the introduction into aliphatic groups, combined with almost water-free conditions, a sufficient solubility in organic solvents and high temperatures, since fluoride is a bad nucleophile. [12] In the case of aromatic nucleophilic substitution, leaving groups like F (isotope exchange), Me 3 N + , NO 2 or other halogens have been used. [13] Tertiary 2-[ 18 F]fluoroethylamine and 3-[ 18 F]fluoropropylamine moieties are structural elements commonly found in several 18 F-radiotracers. The four most commonly used and well-established approaches for the preparation of such radiotracers are illustrated in Scheme 1. The first approach comprises the preparation of a [ 18 F]fluoropropyl or -ethyl moiety (e. g., mostly 1-bromo-3-[ 18 F]fluoropropane or 2-[ 18 F]fluoroethyl tosylate), [14] followed by subsequent introduction of this 18 F-building block into secondary amine precursors using a nucleophilic displacement reaction. Overall radiochemical yields (RCYs) ranging from 10 % to 51 % were obtained using this two-step reaction. [15][16][17][18][19][20] In the second approach, a tertiary amine with an ethyl or propyl residue, equipped with a leaving group at the terminal carbon atom, is directly reacted with [ 18 F]fluoride. Using tosylate or mesylate as leaving groups led to RCYs ranging from 33 % to 70 %, [15,[21][22][23][24][25][26][27] whereas a RCY of 88 % was reported when bromine was employed as the leaving group. [28] A third approach centres around deoxy(radio)-fluorinations which are also known for the introduction of fluorine-18 via a direct replacement of OH groups using, for example, 18 F-versions of DAST (diethylaminosulfur trifluoride), deoxo-fluor (bis(2-methoxyethyl)aminosulfur trifluoride) or PyFluor (2-pyridinesulfonyl fluoride). [29][30][31][32] The fourth approach consists of the application of strained cyclic ammonium salts or small cyclic amines (3-/4-membered rings) as precursors. The reactions of nucleophiles with aziridines or aziridinium ions (3-membered rings), causing the ring opening of the substituted heterocycles, are reported to be facile, [33] especially for the introduction of fluorine-18. However, only little is known about ring-opening reactions of azetidinium salts (4-membered rings). These reactions have been reported to lead to unbranched propyl chains when the heterocycle is unsubstituted. [34][35][36][37][38] Derivatives of αand β-amino acids bearing a fluorine atom at the vicinal aliphatic position have gained widespread applications, for instance in peptide/protein chemistry combined with protein recognition. They represent an important class of enzyme inhibitors, antitumor and antibacterial agents, samples for 19 F NMR analyses and radiotracers for PET when radiolabelled with fluorine-18. [39][40][41]

Aziridines and Aziridinium Salts
Aziridines and aziridinium salts are three-membered heterocyclic compounds with one nitrogen atom in the ring, which, in the case of aziridinium salts, is positively charged. A nucleophilic attack is possible at both carbon atoms of the ring systems, leading to an opening of the strained three-membered ring [42,43] and, in the case of using fluoride as nucleophile, to the formation of a 2-fluoroethyl moiety (Scheme 2).
Aziridines were first used for the synthesis of 18 F-labelled 2fluoroethyl-nitrosourea derivatives. [44] These compounds were reported for the treatment of patients with malignant brain tumours, including brain gliomas. [45,46] 1,1'-Carbonylbisaziridine (1) or N-(2-fluoroethyl)aziridine-1-carboxamide (2) were used for the preparation of [ 18 F]BFNU (Scheme 3). The radiolabelling was performed in acetonitrile at 145°C for 20 min in the presence of 18-crown-6. In the case of using the diaziridine precursor 1, HF was added afterwards to enforce the ring opening of the second aziridine. The nitration of [ 18 F]3 in the last step was conducted in formic acid as solvent with sodium nitrite at 0°C for 5 min, followed by a purification via HPLC with RCYs ranging from 5 % to 10 % for [ 18 F]BFNU.
Additionally, the second radiotracer [ 18 F]CFNU was obtained as mixture of regioisomers after radiolabelling (Scheme 4) with RCYs ranging from 8 % to 15 %. Here, either 1,1'-carbonylbisaziridine (1) or N-(2-chloroethyl)aziridine-1-carboxamide (5) were used as starting materials. The radiofluorination was performed under the above-mentioned conditions also used for [ 18 F]BFNU. For the diaziridine precursor 1, HCl was used to ring-open the second aziridine moiety in the second step after labelling. The last step of both routes involved the nitration of [ 18 F]4 and [ 18 F]6, respectively, as described above, leading to an isomeric mixture of [ 18 F]CFNU after HPLC purification.
In 2000, Trón and colleagues synthesised 18 F-labelled adenosine [ 18 F]13 from the isopropylidene protected aziridine precursor 7 in two steps (Scheme 5). [47] The precursor 7 was obtained from uronic acid 8 which was reacted with aziridine and N,N-diisopropylcarbodiimide as the coupling agent. The nonradioactive reference compound was prepared from uronic acid 8 and 2-fluoroethylamine. The radiolabelling was accomplished with aziridine precursor 7, which was treated with  13 was achieved under acidic conditions and in quantitative yields. The radiolabelling procedure as well as the alternative way are shown in Scheme 5.
Both Cbz-protected regioisomers [ 18 F]15 a,b were subsequently purified using an SPE (solid-phase extraction) cartridge prior to the quantitative deprotection with Pd/C-H 2 to yield Unsymmetrically functionalised aziridine-2-carboxamides are the basis for the direct nucleophilic introduction of fluorine-18 in a regioselective manner, yielding α-amino-β-[ 18 F]fluoropropanamide derivatives (Scheme 7). [50] Four model compounds 18 a-d were chosen to point out the labelling conditions and the influence of the functional group connected to the nitrogen atom of the aziridine. Electron-withdrawing sulfonyl groups have been found superior for the fluorine introduction at elevated temperatures in contrast to the benzoyl group. The radiofluorination experiments were carried out with 2 mg of the respective precursor in DMSO and K 2 CO 3 or Cs 2 CO 3 as base at 70°C for 15  obtained independently of the (radio-)fluorination method, but with a tendency to form the isomer with the lesser steric demand.
To expand the scope, this method was extended to a larger number of compounds and was applied to the radiofluorination of aziridinium salts obtained from β-hydroxyethylamines to yield N,N-disubstituted 2-[ 18 F]fluoroethylamines (Scheme 9). Due to the symmetric unsubstituted aziridinium ring, the ring opening led to a single radiofluorinated product. Three model compounds 27 a-c were treated with [ 18 F]fluoride using the labelling methods described above to obtain the desired products [ 18 F]29 a-c in RCYs of 14 % to 31 %.
The respective nonradioactive reference compounds were prepared using the same precursors 27 a-c, which were first converted into the aziridinium derivatives 28 a-c with trifluoromethanesulfonic anhydride and DIPEA as base. Next, the cyclic intermediates were treated with TBAF to obtain the desired fluoro compounds after chromatographic purification.
In 2015, the same group expanded their portfolio to label various N,N-disubstituted-β-aminoalcohols (Scheme 10) using the one-pot method described above. [52] They investigated the influence of the temperature on the radiochemical yield and on the ratio of the formed regioisomers and found that radiochemical yields varied depending on the substituent connected to the nitrogen atom. In most cases and as expected, the RCY increased with higher reaction temperature. However, when propargyl was used as a substituent, exemplarily shown with precursor 30, a degradation of the 2-fluoropropan-1-amine isomer  The stability of the product depends on the substituents, and the following order was established for the radiolabelling procedure at 90°C: Furthermore, this method was applied to the preparation of [ 18 F]FECNT, a radiopharmaceutical used to image dopamine transporters in the brain. [53,54] The alkylation of the nortropane precursor 33 using different 2-[ 18 F]fluoroethylsulfonates [ 18 F]34 a-c [55][56][57] or bromide is conducted in a two-step procedure with overall RCYs ranging from 16.5 % to 40 % (d.c.) or, alternatively, in one step from the respective mesylate precursor 35, [58,59] but this precursor 35 has been described to be unstable. As an alternative, this working group reported a one-pot procedure starting from the respective aminoalcohol 36, which was first transferred into the aziridinium triflate intermediate 37 and then subsequently radiolabelled using the above-mentioned conditions to obtain [ 18 F]FECNT in a RCY of 12 % (rt) or 21 % (90°C). All three routes are detailed in Scheme 11.
Methyl aziridine-2-carboxylate was equipped with five different electron withdrawing protecting groups (Ts, Ns, Boc, Fmoc, Cbz) for the preparation of α-[ 18 F]fluoro-β-alanine or β-[ 18 F]fluoroalanine. [60] First of all, the 18 F-introduction by ring opening with [ 18 F]fluoride was only successful for derivatives containing Boc, Cbz or Ts as activating group (exemplarily shown for precursor 38). For these precursors, labelling conditions were optimised by varying the solvent, temperature (including microwave conditions), precursor amount, base, and time. Additionally, an HPLC procedure was established to separate both regioisomers. The highest 18 F-incorporation with a RCY of 45 % was found using temperatures � 100°C combined with microwave support and TEAHCO 3 as mild base, furthermore using DMSO as solvent. Notably, the methyl ester of [ 18 F]39 a,b was partially cleaved during the labelling procedure, whereas the activating group was only cleaved after labelling under strong acidic conditions together with the remaining ester to yield the desired radiotracers [ 18 F]40 a,b.
α-[ 18 F]Fluoro-β-alanine [ 18 F]40 a was exclusively obtained after the ring-opening for labelling of the Boc-and Cbzactivated aziridines and after ester hydrolysis and activation group removal (Scheme 12). The aziridine was attacked by [ 18 F]fluoride exclusively at the most substituted α-carbon atom. No other regioisomer was detected. Interestingly, reports on ring opening using the nonactivated isopropyl aziridine-2carboxylate, for example with HF/pyridine, showed completely opposite regioselectivity with attack at the unsubstituted βcarbon atom. [61,62] Another convenient access to [ 18 F]fluoroaminoesters under mild conditions by deoxyradiofluorination of β-hydroxy-αaminoesters derived from serine, α-methyl-serine, or β-phenylserine was described in 2019. [63,64] For this purpose, a three-step procedure was developed, starting from differently N,N-alkylated compounds 41 a-h (R' and R'' = Bn, DMB, Me, propargyl), which were prepared and first treated with triflic anhydride in dichloromethane for 1 h at rt. Next, DIPEA in acetonitrile was added as base to generate the respective aziridinium precursor intermediates 42 a-h. Afterwards, K[ 18 F]F-K222 and K 2 CO 3 as base in acetonitrile were added and the final mixture was stirred at rt for 30 min. This subsequent radiofluorination, under optimised conditions, yielded the desired 18

F-radiotracers [ 18 F]43 a-h and [ 18 F]44 a-h as isomeric mixture in different ratios and in RCYs ranging from 10 % to 75 % (Scheme 13).
In principle, two regioisomers are possible from radiofluorination by nucleophilic attack of the [ 18 F]fluoride due to the two differently substituted carbon atoms of the three-membered ring. Notably, both isomers were found when using phenylserine 41 g,h (R=H and R''' = Ph) with the α-isomer [ 18 F]43 g,h dominating. In contrast, the radiofluorination is completely regioselective when using serine or methylserine derivatives 41 a,b,d,e (R=H or Me and R''' = H). Only the respective β-aminoesters [ 18 F]44 a,b,d,e were obtained. No radiolabelled product was found when using the dimethoxybenzyl group (R' = DMB) for amine protection as found in precursors 41 c,f, probably due to the steric hinderance of this bulky group.
The appropriate reference compounds were prepared using either the direct deoxyfluorination with DAST or the procedure with the aziridinium precursors and TBAF as fluorine source, also yielding both regioisomers for R=H and R''' = Ph and, in the

Azetidinium Salts
Azetidinium salts are organic four-membered heterocyclic compounds with one positively-charged nitrogen atom in the ring. They are sensitive to nucleophilic attacks at ring positions C-2 and C-4 due to the positive charge of the nitrogen atom that leads to a S n 2 reaction yielding the respective ring-opened product. [65] Of all small cyclic compounds, the four-membered representatives are generally the most difficult to synthesise. However, a number of new synthetic methods have been elaborated, [66][67][68][69] including the enantioselective nucleophilic ring opening of these salts by fluoride, leading to γ-fluoropropylamines (Scheme 14). [70] The first investigation to introduce fluorine-18 via azetidinium precursors was reported in 2004 by Kiesewetter and Eckelman. [71] They aimed to prepare 4-{[1-(3-[ 18 F]fluoropropyl)-4piperidinyl]-methoxy}benzonitrile ([ 18 F]48), a 18 F-radiotracer to image the sigma1 receptor with high selectivity. [72] During the synthesis of the precursor 47, they found that the open-chain mesylate derivative 46 spontaneously cyclised, forming the respective azetidinium salt. In-depth NMR investigations were carried out to elucidate the structure of the azetidinium salt. The following radiolabelling reaction was performed in acetonitrile, using K 2 CO 3 as base and the azeotropically dried  18 F-incorporation into the azetidinium salts even at rt or slightly elevated temperatures.
Interestingly, the respective nonradioactive reference compounds were not prepared from the spiro precursors, instead 1bromo-3-fluoropropane and the appropriate secondary amines have been used for this purpose.
In 2011, Mamat et al. used 1-(3-[ 18 F]fluoropropyl)piperazines as model compounds to prepare 18 F-radiotracers based on inhibitors to image cyclin-dependent kinases (CDK), [73] since the piperazine moiety is a basic structural element of these CDKinhibitors (Scheme 16). [74] They started with 1-(4nitrophenyl)piperazine 53 a and 1-(6-nitropyridin-3-yl)piperazine 53 b, respectively, which were treated with 3-bromopropanol to introduce the 3-hydroxypropyl chain. After tosylation of the alcohol functions of 54 a,b, the open-chain derivatives 55 a,b were obtained, which tend to cyclise spontaneously to the spiro salts 56 a,b even at room temperature and much better in polar solvents like DMSO. Both azetidinium precursors 56 a,b were then treated with the azeotropically dried K[ 18 F]F-K222-complex at 80°C for 30 min, yielding both 18 F-compounds [ 18 F]57 a,b in RCYs > 88 %. Interestingly, a high RCC of > 65 % was observed for both spiro precursors even after 5 min at 80°C.
In 2013, this method was transferred, by the same group, to prepare two radiolabelling building blocks [ 18 F]AFP and [ 18 F]BFP for bioorthogonal 18 F-introduction, [75] which can be used for labelling purposes either with the traceless Staudinger Ligation [76] or using Cu-mediated or -free click reactions. [77,78] Based on the piperazine skeleton, the respective reference compounds AFP and BFP as well as the spiro precursors 60 a,b were synthesised by first introducing the azide or alkyne function into the piperazine moiety yielding 58 a,b (Scheme 17). The next step involved the alkylation with 3-bromopropanol to alcohols 59 a,b, followed by subsequent tosylation with tosyl chloride. Both open-chain tosylates were subsequently purified by means of column chromatography and then heated in methanol to induce cyclisation to spiro precursors 60 a,b (43 % yield for both).
NMR analyses, MS and XRD confirmed the molecular structure of the formed spiro salts 60 a,b. As one example, XRD analysis elucidated and confirmed the conformation of the two rings connected by the central nitrogen atom of BFP precursor 60 b with a tosylate counterion, showing a chair conformation of the 6-membered ring as shown in Figure 1. [79] The subsequent radiolabelling was accomplished with the spiro precursors 60 a,b, which were treated with K[ 18 F]F-K222 and K 2 CO 3 as base in acetonitrile at 100°C for 15 min with nearly quantitative conversion of [ 18 F]fluoride (start activity 1-12 GBq). Advantageously, a simple silica gel cartridge could be used for purification (Figure 2) of both radiolabelling building blocks [ 18 F]AFP and [ 18 F]BFP, leading to RCPs > 97 %. Finally, an automated module synthesis was developed, yielding both building blocks in approx. 30 % RCY. [80,81] In a proof-of-concept study, different azide-functionalised amino acids were radiolabelled with these building blocks. Interestingly, it was possible to radiolabel small molecules like alkyne-functionalised amino acids with [ 18 F]BFP using Cumediated click-radiolabelling reactions. However, this building block was not found suitable with alkyne-functionalised peptides [81] due to the formation of bis-alkynes via the Glaser coupling. [82] Instead, [ 18 F]AFP was used for the radiolabelling of the SNEW peptide [ 18 F]62, [83] which is known to be a substrate for the EphB4 receptor, and for radiolabelling the SWLAY peptide [ 18 F]61, [84] (Scheme 18) which is known to be a substrate for the EphA2 receptor. [85] A high-affinity EphB4 receptor ligand (box in Scheme 19) published by Bardelle et al. [86] was used as basis for the development of potential PET radiotracers with fluorine-18 and carbon-11. [87,88] The position for the introduction of carbon-11 was easy to find because of the prominent methyl group in the molecule, allowing for the use of [ 11 C]methyl iodide for radiolabelling.  In silico methods were used to investigate the suitability of different positions in the native molecule prior to the introduction of [ 18 F]fluoride, aiming to prevent a loss of affinity to the EphB4 receptor. These studies resulted in the identification of two 18  The PET-radiotracer N-3-[ 18 F]fluoropropyl-2-β-carboxymethoxy-3-β-(4-iodophenyl)nortropane, also known as [ 18 F]FP-CIT, is a very prominent example for illustrating different strategies to obtain the [ 18 F]fluoropropyl moiety. The cocaine analogue FP-CIT [89] has been described as a dopamine transporter ligand, and the radiolabelled compound [ 18 F]FP-CIT, together with [ 123 I]FP-CIT, [90] are widely used for the detection or exclusion of nigrostriatal degeneration in patients with clinically uncertain Parkinsonian syndrome. [91,92] Different radiofluorination methods using 3-[ 18 F]fluoropropyl tosylate (25.3 � 2.1 %), [93,94] 3-[ 18 F]fluoropropyl triflate (RCY 70 %), [95] or 3-[ 18 F]fluoropropylbromide (RCYs 2-49 %) [19,96,97] in a two-step procedure with 68, or the direct way either from the open-chain precursor 67 [28,98] or the azetidinium precursor 71 (RCY 92.4 � 3.6 %) [99] have been described (Scheme 20), varying not only the solvent, the amount of precursor, the reaction time and the solvent.
The highest RCY of [ 18 F]FP-CIT was found when the respective azetidinium salt 71 a,b was used as precursor, furthermore employing methyl 2-hydroxyisobutyrate as solvent in a one-pot reaction (Scheme 21). For this purpose, the precursor was prepared from cocaine skeleton 68 (also known as nor-β-CIT), which was treated with 3-bromopropanol to yield 69 [100] and afterwards sulfonylated with Ts 2 O or Ms 2 O, respectively, to obtain the open-chain precursors 70 a,b. [101] The cyclisation to the resulting precursors 71 a,b was induced by heating in polar solvents. Interestingly, the ring opening was achieved after 1 h at 70°C, using benzene as nonpolar solvent. The fluorination was accomplished with TBAF in t-BuOH at 80°C. After 30 min, the nonradioactive reference compound FP-CIT was obtained in a yield of 53 %. The respective radiolabelling was described for the open-chain compound, leading to a decay-corrected radiochemical yield of 35.8 � 5.2 % in an automated module synthesis. [102] The respective mesyl spiro precursor 71 a is commercially available.
The combination of using azetidinium salts as precursors and the minimalist approach for radiofluorinations was highlighted in a work of Omrane. [103] This approach is based on the use of ammonium salts as precursors to avoid phase transfer catalysts and additives like Kryptofix K222 for the radiolabelling process with fluorine-18. [104] spiro-Ammonium salts are therefore the ideal precursors. For this purpose, 7-benzyl-2-hydroxy-4azaspiro [3,5]nonan-4-ium p-tosylate (74) was prepared according to a procedure published by Sladowska and co-workers from 72 and 73. [105] To perform the radiofluorination, [ 18 F]fluoride was trapped on an anion-exchange cartridge and eluted with the precursors 74 or 75, respectively, dissolved in methanol. After changing the solvent to acetonitrile, the mixture was heated to 110°C for 15 min. The initial experiments were performed with chloride as anion (precursor 74); leading to a radioactive by-product in high yields (38 %). After changing to tosylate as counterion (precursor 75), the desired radiofluorinated compound [ 18 F]76 was obtained in up to 84 % when using acetonitrile as solvent at 110°C for a reaction time of 15 min (Scheme 22). The respective reference compound was prepared in 51 % yield using TBAF(t-BuOH) 4 in acetonitrile at 75°C after 4 h. Additionally, this method was transferred to the production of [ 18 F]AFP from the respective spiro precursor 60 a. A RCC of 94 % was observed under the above-mentioned optimised conditions.

Conclusion
The use of aziridines, their aziridinium salts, and azetidinium salts represents a convenient access to obtain pharmacologically interesting compounds like β-amino acids with either 2fluoroethylamino or 3-fluoropropylamino moieties, respectively, as well as radiotracers with the corresponding 2-[ 18 F]fluoroethylamino or 3-[ 18 F]fluoropropylamino pattern, which were available mostly in high radiochemical yields and purities. In many cases, the respective precursors are readily accessible and the following radiofluorination proceeds smoothly under mild labelling conditions with high RCCs due to the anchimeric assistance of the nitrogen in βor γ-position. More stringent labelling conditions are required for the nucleophilic 18 Fintroduction into aziridines. Furthermore, regioselectivity depends on sterically demanding functional groups. Additionally, methods and strategies were elaborated for a regioselective introduction of fluorine-18 when using unsymmetrically substituted ring systems by tuning the reaction conditions and varying the substituents at the ring system.